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A comparative proteomics approach to detect unintended effects in transgenic Arabidopsis

Journal of Genetics and Genomics 2009年第10期36卷 629-639页

作者： Yanfei Ren Jun Lv Hua Wang Linchuan Li Yufa Peng Li-Jia Qu National Key Laboratory of Protein Engineering and Plant Genetic Engineering College of Life Sciences Peking University Beijing 100871 China lnstitute of Plant Protection Chinese Academy of Agricultural Sciences Beijing 100193 China

Recently, as part of biosafety assessments, unintended effects have been given much attention. In this study, we applied a proteomics approach to elucidate the unintended effects of random T-dNA insertion in transgenic plants. Separated proteins extracted from 12 transgenic Arabidopsis thaliana with different T-dNA insertion sites and from wild-type （ecotype Col-o） were analyzed. In the transgenic plants, 102 significantly altered protein spots were detected, in which 59 were up-regulated and 43 down-regulated. MALdI-TOF MS analysis showed that most of these expression level-altered proteins were involved in energy transfer, oxidative respiration and photosynthesis. However, none of these proteins was a toxic protein or allergen. Using plants with or without cold treatment, a natural environmental stress, as controls, we found that the number of the altered proteins was even less in those transgenic plants than those triggered by the cold treatment, suggesting that the transgenic events had a weaker impact on the plants than the environmental stresses. Interestingly, the phosphinothricin acetyl transferase （PAT）, the BAR-encoded protein, was detected in nine out of twelve different T-dNA insertion lines at five different insertion sites. These data suggest that the most significant impact of transgenic events on the host plants is from the transgene itself, i.e., from the predictable intended effects, rather than unintended effects. This study also suggests that the proteomics approach has the potential to detect the unintended effects in transgenic plants.

关键词： biosafety proteomics Arabidopsis thaliana 2-d page MALdI-TOF unintended effect

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Proteomic Comparison of Two-dimensional Gel Electrophoresis Profiles from Human Lung Squamous Carcinoma and Normal Bronchial Epithelial Tissues

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Genomics, Proteomics & Bioinformatics 2003年第1期1卷 58-67页

作者： CuiLi XianquanZhan MaoyuLi XiaoyingWu FengLi JianlingLi ZhiqiangXiao ZhuchuChen XuepingFeng PingChen JingyunXie SongpingLiang MedicalResearchCenter XiangyaHospitalCentralSouthUniversityChangsha410008China CancerResearchInstitute XiangyaSchoolofMedicineCentralSouthUniversityChangsha410078China CollegeofLifeScience HunanNormalUniversityChangsha410006China

differential proteome profiles of human lung squamous carcinoma tissue compared to paired tumor-adjacent normal bronchial epithelial tissue were established and analyzed by means of immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis (2-d page) and matrix-assisted laser desorp-tion/ionization time of flight mass spectrometry (MALdI-TOF-MS). The results showed that well-resolved, reproducible 2-dE patterns of human lung squamous carcinoma and adjacent normal bronchial epithelial tissues were obtained under the condition of 0.75-mg protein-load. The average deviation of spot position was 0.733±0.101 mm in IEF direction, and 0.925±0.207 mm in SdS-page direction. For tumor tissue, a total of 1241±88 spots were detected, 987±65 spots were matched with an average matching rate of 79.5%. For control, a total of 1190±72 spots were detected, and 875±48 spots were matched with an average matching rate of 73.5%. A total of 864±34 spots were matched between tumors and controls. Forty-three differential proteins were characterized: some proteins were related to oncogenes, and others involved in the regulation of cell cycle and signal transduc-tion. It is suggested that the differential proteomic approach is valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis. These data will be used to establish human lung cancer proteome database to further study human lung squamous carcinoma.

关键词： human lung squamous carcinoma tissue normal bronchial epithelial tissue 2-d page MALdI-TOF-MS proteome

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A Comparative Analysis of the Endosperm Proteins Separated by Two-dimensional Electrophoresis for two Cultivars of hybrid Rice (Oryza saliva)

A Comparative Analysis of the Endosperm Proteins Separated b...

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首届全国蛋白质组学学术研讨会

作者： Yang Pingfang Shen Shihua Kuang Tingyun Key laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China Graduate College of Chinese Academy of Sciences, Beijing 100039, China

Liangyoupeijiu and Shanyou63 are two parental and three parental lines hybrid rice, respectively. Although both of them belong to indica subspecies, they have obvious differences in morphology, physiology and grain quality. Variations in endosperm protein compositions between these two cultivars of hybrid rice were analyzed by two dimensional gel electrophoresis (2-d page) in our study. After matrix assisted laser desorption/ionization time of flight mass spectrometry (MALdI-TOF MS) analysis, a 21 Kda precursor of 19 Kda globulin was identified as the major storage protein for both the cultivars. Some isoforms of peroxiredoxin and seed maturation protein were detected only in Shanyou63, while aldose reductase and starch granule-bound starch synthase can be detected only in Liangyoupeijiu. These proteins may contribute to the differences in grain quality between the two cultivars of hybrid rice.

关键词： Oryza sativa Endosperm Hybrid rice 2-d page MALdI-TOF MS

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蛋白质组学方法学研究与水稻花药蛋白质组的初步分析

蛋白质组学方法学研究与水稻花药蛋白质组的初步分析

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作者：李小兰湖南师范大学

学位级别：硕士

蛋白质组是指一个基因组所编码的所有蛋白质的总和。蛋白质组方法学包括蛋白质组分离技术和蛋白质组鉴定技术。用于蛋白质分离的双向凝胶电泳技术是蛋白质组研究的核心，而鉴定技术是蛋白质组技术的基础。本文以虎纹捕鸟蛛(selenoc... 详细信息

蛋白质组是指一个基因组所编码的所有蛋白质的总和。蛋白质组方法学包括蛋白质组分离技术和蛋白质组鉴定技术。用于蛋白质分离的双向凝胶电泳技术是蛋白质组研究的核心，而鉴定技术是蛋白质组技术的基础。本文以虎纹捕鸟蛛(selenocosmia huwena)粗毒为材料，对影响双向凝胶电泳结果的关键因素与环节予以优化，如样品处理、电泳参数和凝胶浓度等。结果表明：采用增溶排异的裂解法处理样品，选择适当的上样量，用预制的固相pH梯度胶-IPG胶条(pH3-10)进行第一向等电聚焦，并选用15％的均一胶进行第二向SdS电泳，可获得质量较好的双向电泳图谱。并以分离粗毒后凝胶为基础，对蛋白质组鉴定技术-图像分析，微量测序，肽质谱指纹图等进行了方法学上的探讨。将双向凝胶电泳后的凝胶印迹转移至PVdF膜上，随机对膜上分子量10kd以下的组分进行了N-端序列测定，鉴定到了虎纹捕鸟蛛毒素HWTX-1、HWTX-11、HWTX-1V和SHL-1在凝胶上的位置，同时发现了5种新的肽类毒素组分。并对双向凝胶电泳后银染显色的蛋白质斑点进行了MALdI-TOF-MS肽质指纹图分析，初步确定了10kd以上6个蛋白质在凝胶上的位置和发现了几种新的肽类组分。以建立的蛋白质组研究技术为基础，对两性系温敏核不育水稻双低-S不同育性条件的减数分裂期花药蛋白质进行了研究，初步建立并优化了水稻蛋白质组分析的双向聚丙烯酰胺凝胶电泳技术，达到了提高其分辨率和重复性的目的。以固相pH梯度等电聚焦为双向电泳的第一向，并优化电泳条件。结果表明：在增溶样品，设定合适的电泳参数，选择适当的SdS-page的凝胶浓度等电泳条件下，能获得质量较好的凝胶图谱。并利用PdQUEST 2-d分析软件对扫描后的凝胶图谱进行了处理，获得了清晰度高的凝胶图谱;用不同域值对蛋白质斑点进行识别，第二章蛋白质组学方法学研究分别得到了1000多个斑点;通过初步的斑点配比，获得了温敏不育条件下与可育条件下花药蛋白质的差异蛋白质点图谱;利用2一d低分子量标准，对不育条件下的图谱进行了等电点与分子量初步确定;并对可育条件下花药蛋白质双向电泳凝胶上的45个蛋白质斑点作了等电聚焦和sdsAGE位置重复性分析:蛋白质斑点位置偏移沿等电聚焦轴为 x.45土0.23mm，沿sdS一page轴为1.15土0.17mm。并对经双向凝胶电泳分离的银染蛋白质点进行了部分点的肤质谱指纹图分析，初步对双向电泳凝胶上不育条件下花药蛋白质的20多个点作了归属。

关键词：双向凝胶电泳蛋白质组蜘蛛毒素微量测序 2-d page 水稻花药肽质谱指纹图 MALdI-TOF MS

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